Two males and two females were assigned randomly to the treatment group and
one male and two females to the control group. Until 11 February, deer were
fed ad libitum a high protein (11.1% crude protein)-high energy (2,990 kcal
digestible energy/kg) pelleted diet (DelGiudice et al. 1990; 1994a).
Baseline data were collected on 4 February during 0800 to 1200 hours. Using
a pole syringe, we anesthetized deer by injection of 100-150 mg xylazine HCl
and 200-650 mg ketamine HCl. Once induced, deer were weighed, and blood and
urine were collected (DelGiudice et al. 1994a).

Treated deer (i.e., restricted diet) were provided with 0.2-1.0 kg/deer/day
of a low protein (7.0% crude protein)-low energy (1,900 kcal digestible energy/kg)
pelleted diet from 11 February to 5 May 1988, except for 15 to 19 April (see
below). From 11 February to 15 April, each control deer received the same
diet ad libitum as the treated deer. We believed ad libitum provision of this
diet to be more realistic for a control group in a winter nutrition study,
as opposed to an unnatural, high-quality diet (DelGiudice et al. 1994a).
To simulate and examine the effect of acute severe nutritional restriction
(as might accompany a snowstorm) on control deer, we restricted all seven
deer to 0.2 kg of feed per day from 15 to 19 April; ad libitum feeding was
resumed for control deer from 19 April (after handling) to 5 May. Overall,
mean daily feed intake was 0.53 kg (95% confidence interval, 0.47-0.59 kg)
and 0.28 kg (95% confidence interval, 0.06-0.50 kg) for the control and restricted
deer, respectively. Maximum cumulative mass loss was greater in restricted
deer (17.0%-32.2%) than in control deer (7.0%-17.4%) (DelGiudice et al. 1994a).
Deer were dependent upon snow for water until late February, when we provided
water ad libitum.

From 11 February to 5 May, we anesthetized and handled all deer as described
above at 1- to 2-week intervals. On average, 10 and 62 minutes elapsed between
induction of anesthesia and blood and urine-sampling, respectively (DelGiudice
et al. 1994a).

Laboratory and statistical analyses

Urinary creatinine was determined spectrophotometrically with an ABA-100
bichromatic autoanalyzer using modifications of the method of Jaffe (1886).
Urinary 3-methylhistidine was analyzed using cationic-exchange resin and spectrophotometry
(Vielma et al. 1981; Fitch et al. 1986). Urinary 3-methylhistidine is expressed
as a ratio to creatinine (µmol:mg) to correct for differences of hydration
and as an estimate of fractional catabolic rate (Haverberg et al. 1975; Coles
1980; Elia et al. 1981; Ballard and Tomas 1983).

We evaluated the temporal pattern of 3-methylhistidine: creatinine in deer
from treatment and control groups by fitting a mixed-effects, repeated measures
analysis of covariance model to the log-transformed data (ANCOVA; Ware 1985;
SAS PROC MIXED, SAS Institute Inc. 1996). The model contained fixed effects
for diet, time, time2, diet x time, and diet x time2;
deer were modeled as random "subject" effects. Using the techniques of Wolfinger
(1993), a first order antedependence covariance structure was selected to
account for the within-deer correlations. A mixed effects, repeated measures,
polynomial regression model was used to evaluate dependence of 3-methylhistidine:creatinine
on percent mass loss of deer. In this case, a compound-symmetry covariance
structure was employed. We fit polynomials of the form: